Understanding information processing in the cerebral cortex requires understanding the role of feedforward (FF) and feedback (FB) circuits between lower and higher cortical centers. Organizing principles for these circuits, that could determine how they process sensory information, remain largely unknown. This is due to the complexity of inter-areal circuits, i.e. their anatomical and functional specificity, and the lack of methodologis that can reveal the fine-scale connectivity of FF and FB circuits made by specific cell types, and relate it to the functional architecture of the cortex. Ultrastructural-scale circuit anatomy, whil useful for building wiring diagrams of local connections in mouse cortex, cannot be used to study the large cortical volumes encompassed by inter-areal axons. The latter can be studied only at mesoscopic scale. Our goal is to develop a methodology for labeling and efficiently reconstructing, single cell types and their inter-areal axons. Previous single axon studies were affected by ambiguity in the origin of the axonal label, inability to restrict label to few neurons and laborious manual reconstructions. These studies have provided only a small sample of incompletely reconstructed axons, biased towards regions of sparser labeling, with no identification of their cell types of origin. Our Specific Aims are: Aim 1. To label unambiguously at high resolution the axon of single projection neurons, and to develop a novel computational framework for semi-automated single axon reconstruction. We will extend viral-mediated expression of GFP to labeling at high resolution, and sparsely the axons of inter-areal projection neurons. We will develop a novel approach for fast serial section reconstruction of single axons, which includes 3D imaging of intact tissue blocks rendered optically-transparent, and novel computational algorithms for semi-automated axon segmentation. Aim 2. To apply these methods to resolve controversies in the literature on the functional specificity, or lack thereof, f inter-areal feedback projections to primate visual cortical area V1. Two previous studies of the layout of V2 FB projections onto the V1 orientation map have demonstrated orientation-specific, one, and unspecific FB connections, the other. Our preliminary data suggest existence of two FB systems, likely related to different cell types, which show unique relationships to the cortical functional architecture, thus providing a way to reconcile apparently contradictory data. The contribution of the proposed research is significant because it will provide new tools for studying the fine-scale connectivity and functional organization of inter-areal circuits made by specific cel types. General organizing principles for these connections will emerge that will provide an anatomical foundation for hypothesis-driven studies of their function. The proposed research is innovative because unlike previous studies: 1) the novel labeling method permits high-resolution, unambiguous identification of single inter-areal neurons, from soma to axon; 2) semi-automated mapping of 3D volumes from serial sections allows for fast reconstruction and, thus, higher yield of reconstructed axons; 3) it combines for the first time functional imaging of corticl responses with labeling of single FB axons. PUBLIC HEALTH RELEVANCE: Normal brain function depends on the orderly development of circuits in the cerebral cortex and on their intact function. Knowledge of the normal circuitry provides a foundation for understanding the causes of impaired brain function and developing corrective measures. Our proposed novel approach for studying the detailed microcircuitry of single long-distance axons can be applied to any area of the cerebral cortex and to any mammalian species, including higher mammalian species with large brains. This tool for studying the detailed inter-areal circuitry in the cerebral cortex can be used for studying the normal circuitry and how the latter is modified in altered brain function. As an example, our studies of the normal circuitry between early visual cortical areas will provide greater insight ino the causes and effects of central vision defects when these circuits are damaged by stroke or other insult. In particular, our studies on feedback circuits between different visual cortical ares will help our understanding of how these pathways mediate the influence of stimulus context on neuronal responsivity, spatial integration, and changes in visual sensitivity associated with top-down attention and learning. Understanding how these circuits operate in normal vision will provide a foundation for understanding the consequences of their dysfunction in abnormal states, such as impaired visual contextual integration in schizophrenia (Dakin et al., 2005; Yoon et al., 2010), visuo-spatial local-to-global interference in autism (Wang et al., 2007; Simmons et al., 2009), as well as disorders of visual attention and learning.